The goal of this project is to understand how proteins which must be secreted or inserted into membranes are identified and directed to transport sites in the endoplasmic reticulum (ER) or, equivalently, the bacterial cytoplasmic membrane. The project combines biochemical and genetic methods to study the role of the signal recognition particle (SRP), the SRP receptor, and their bacterial homologs in this process. Previous studies have shown that the SRP ribonucleoprotein (RNP) recognizes nascent polypeptide chains bearing "signal sequences" and then catalyzes their translocation across the membrane upon interaction with the ER-bound SRP receptor. A major aim of this project is to elucidate the mechanism by which SRP recognizes signal sequences and releases them in a regulated manner. By studying this problem we expect to obtain insight into the structural features that enable a protein to possess broad substrate specificity and into the regulation of multi-step pathways. Previous studies have shown that signal sequences bind to a C-terminal proteolytic fragment ("M- domain") of the 54kd subunit of mammalian SRP (SRP54). An N-terminal fragment that consists of about 100 amino acid segment ("N-domain") followed by a GTPase module ("GTPase-domain"), however, also contributes to signal sequence binding. We have recently found that the N-domain moeity of this fragment plays an important role in signal sequence binding. Mutations in a conserved motif in the N-domain ("ALLEADV") produce defects in signal sequence binding that correlate with the severity of the mutation. The magnitude of the defect is independent of the preprotein substrate, suggesting that the mutations affect only the affinity of signal sequence binding. The N-domain mutants also show defects in promoting the translocation of presecretory proteins across the membrane of microsomal vesicles, but these defects are a direct consequence of the reduction in signal sequence binding activity and not separate effects of the mutations. By contrast, mutations in the GTPase consensus sequence have no effect on signal sequence binding, but instead severely impair protein translocation activity. Our data are consistent with a model in which conformational changes driven by the SRP54 GTPase cycle regulate the binding and release of signal sequences, possibly by modulating the relative orientation of the N- and M-domains.Although gram- negative bacteria contain an SRP-like RNP comprised of a protein ("Ffh") and a small RNA ("4.5S RNA"), the function of this particle is not yet clear. We have recently obtained substantial evidence that the Ffh/4.5S RNA particle interacts with nascent polypeptide chains at a specific step of the ribosome cycle and targets them to the previously-characterized SecY translocation complex. Furthermore, using a novel genetic method we have identified a set of proteins which may be Ffh/4.5S RNA targeting substrates.